In the United States and throughout the world, cancer incidence and mortality has increased dramatically in both developed and developing nations. Cancer causes ~13% of human deaths with 7.6 million people dying from cancer in 2007. More people in the US die of lung cancer than breast, colon, kidney, and prostate cancers combined. Recent studies show that veterans are 25 to 75 percent more likely to develop lung cancer than people who did not serve in the military. Advances in cancer immunotherapy are leading to breakthroughs in treatment. Adoptive transfer of T cells expressing chimeric antigen receptors (CAR-T) results in durable remis- sions for B cell malignancies. Checkpoint blockade with antibodies against PD-1, PD-L1, and CTLA-4 results in partial and complete responses in patients with a variety of malignancies. Yet, significant limitations exist. With the exception of patients with melanoma, only a minority of patients respond to checkpoint blockage. Common cancers such as prostate and colorectal cancer generally do not respond. Thus, additional approaches are needed to realize the full potential of cancer immunotherapy. Treatment with ?? T cells expressing V?2V?2 TCRs is one such approach. Unlike ?? T cells, the response of V?2V?2 T cells is not MHC restricted but instead requires the Ig superfamily protein, butyrophilin 3A1, to sense the foreign-microbial isoprenoid metabolite, HMBPP, and the self-metabolite, IPP. This sensing allows tumor cells to be recognized and killed by V?2V?2 T cells independent of their mutational burden. V?2V?2 T cells safely expand to very high numbers during many infections (up to 1 in 2 circulating T cells) where they kill infected cells and secrete inflammatory Th1 cytokines, chemokines, and growth factors. Two approaches are being used to treat cancer with V?2V?2 T cells. The first is to immunize with stimula- tors such as the bromohydrin analog of HMBPP or the aminobisphosphonate zoledronic acid with low-dose IL- 2. Although treatment has resulted in partial remissions, these vaccines eventually cause anergy and deletion of the V?2V?2 T cells. The second is to adoptively transfer V?2V?2 T cells. This approach is safe and has in- duced complete remissions in three patients with solid tumors, and induced partial remissions or stable dis- ease in others. However, for widespread adoption, V?2V?2 T cell therapy needs to be more effective. Live bacterial vaccines have been used to prevent tuberculosis, typhoid fever, and tularemia. The bacteria produce compounds that activate innate immunity and antigens that stimulate ?? T cells to provide help to the V?2V?2 T cells as they expand. We have now identified an attenuated Listeria strain that consistently ex- pands V?2V?2 T cells. Listeria preferentially accumulate in tumors which should allow the specific tar- geting of adoptively-transferred V?2V?2 T cells to tumors as well as TCR stimulation at the tumor site by HMBPP. We also find that V?2V?2 T cells rapidly express checkpoint receptors such as CTLA-4, PD- 1, TIM-3, and LAG-3 upon stimulation and that adding PD-1 checkpoint blockade markedly enhances prostate tumor immunity by V?2V?2 T cells in a mouse model. We have also identified a novel bisphosphonate prodrug. To accomplish these goals, we will: delete inlB in ?actA prfA* (G155S) Listeria vaccine and assess dosing and timing of immunization, assess the ability of Listeria bacteria to target and acti- vate adoptively transferred V?2V?2 T cells to control tumors, and assess effectiveness of combining checkpoint blockade and a new PTA bisphosphonate prodrug with adoptively transferred V?2V?2 T cells. We have an outstanding team with an excellent track record and have extensive experience working with ?? T cells and isoprenoid metabolism. We have established in vivo models and techniques. An effective Liste- ria vaccine have been identified. The molecular methods to create more vaccines are well developed. In con- clusion, immunotherapy using bacterial vaccines for V?2V?2 T cells has the potential to be broadly applicable for the treatment of many different tumors both by direct activation and through potentiating adoptive transfer.